Proteomic assay using quantum sensors

ABSTRACT

Apparatus and methods for the detection of proteins in biological fluids such as urine using a label-free assay is described. Specific proteins are detected by their binding to highly specific capture reagents such as SOMAmers that are attached to the surface of a substrate. Changes to these capture reagents and their local environment upon protein binding modify the behavior of color centers (e.g., fluorescence, ionization state, spin state, etc.) embedded in the substrate beneath the bound capture reagents. These changes can be read out, for example, optically or electrically, for an individual color center or as an average response of many color centers.

FIELD

The present invention relates to the simultaneous detection of hundredsor thousands of proteins in biological fluids such as blood or urine, afirst step in proteomic analysis. In particular, the present inventionachieves such detection without resorting to mass spectrometry orchromatographic methods, and utilizes a label-free assay, allowing forinstrumentation that is compact, inexpensive, and reusable.

INTRODUCTION

Conventionally, various attempts to evaluate genetic activity or decodebiological processes, including disease process or a biological processof pharmacological effect, have been focused on genomics. However,proteomics can provide further information about the biological functionof cells and organisms. Proteomics includes qualitative and quantitativemeasurement of gene activity by detecting and quantifying the expressionon a protein level rather than the genetic level. Proteomics alsoincludes a study of events which are not coded genetically, such as apost-translational modification of proteins and interactions betweenproteins.

At present, it is possible to obtain an enormous volume of genomeinformation. DNA chips have come into practical use as molecular arraysfor this purpose and the price of direct DNA sequencing has continued todrop significantly. Likewise, there is an increasing demand for highthroughput proteomics. In order to detect proteins, which are morecomplicated and more variable in biological functions than DNA, thereare proposed protein chips, which are currently the subject of intensestudy for many applications. Proteomics is far preferable to genomicsfor the monitoring of health, as the genome is static, indicating onlymedical potential, while the proteome varies dynamically with apatient's medical state, and may even be said to define their medicalstate. However, detecting and quantitating proteins is hard, whiledetecting and quantitating nucleic acids is relatively easy. This hasmotivated many efforts to measure mRNA (messenger RNA) concentrations asa proxy for protein concentrations. Unfortunately, mRNA concentrationshave been shown not to correlate well with protein concentrations. Itappears that proteomics necessarily relies on the ability to detectproteins directly.

“Protein chip” is a collective term used to refer to any device in whichprotein or a molecule for catching such a protein (a capture reagent) isfixed on a surface of a chip, allowing for the detection of proteinbinding. Until recently, the capture reagents on protein chips wereoverwhelmingly antibodies. Detection of specific proteins in complexmixtures such as biological fluids demands high specificity, so manyprotein chips utilizing antibodies also depend on sandwich assays inorder to boost specificity. Such assays are known to have significantshortcomings for proteomics, some of which are protein and/or antibodyspecific and some of which are specific to sandwich assays.

At present, there is no economically viable means by which proteomicdata may be collected from human subjects on a routine basis. Proteomicmeasurement of samples is usually accomplished by variouschromatographic techniques for sample preparation combined with variousmass spectrographic techniques for detection and quantification. Theinstruments used for these procedures are both costly and bulky, so thatsamples usually must be shipped to a central processing facility. Theneed for sample transport requires that samples be processed at thepoint of collection for storage during shipping, or stored on site untiltransport is available. Unfortunately, preparation and storage protocolstend to vary widely across sites, and even within the same sites.Differences in these protocols invariably lead to significant variationin the downstream proteomic measurements, rendering analysis difficultor impossible.

An ideal proteomic collection device would minimize cost by approachingfully solid-state operation (few moving parts), utilizing label-freedetection techniques so that reagent use would be minimal, would bereusable for an indefinite number of measurement runs, and would operateon a small volume of biological fluid. Variations in sample analysis dueto variation in sample preparation and storage protocols could bereduced by minimizing or eliminating sample preparation, and byperforming sample measurement at the place and time and collection,eliminating sample storage and transport.

A new class of non-protein-based capture reagents is found in nucleicacid molecules. The dogma for many years was that nucleic acids hadprimarily an informational role. Through a method known as “SystematicEvolution of Ligands by EXponential enrichment,” sometimes termed theSELEX process, it has become clear that nucleic acids have threedimensional structural diversity not unlike proteins. The SELEX processis a method for the in vitro evolution of nucleic acid molecules withhighly specific binding to target molecules and is described in U.S.patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled“Systematic Evolution of Ligands by EXponential Enrichment,” nowabandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”, U.S.Pat. No. 5,270,163 (see also WO91/19813) entitled “Nucleic Acid Ligands”each of which is hereby incorporated by reference into the presentdisclosure. Each of these publications, collectively referred to hereinas the SELEX Patent Applications, describes a method for making anucleic acid capture reagent to any desired target molecule.

The SELEX process provides a class of products which are referred to asnucleic acid ligands or aptamers, each having a unique sequence, andhaving the property of binding specifically to a desired target compoundor molecule. Each SELEX-identified nucleic acid capture reagent is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets.

The SELEX method applied to the application of high affinity bindinginvolves selection from a mixture of candidate oligonucleotides andstep-wise iterations of binding, partitioning and amplification, usingthe same general selection scheme, to achieve virtually any desiredcriterion of binding affinity and selectivity. Starting from a mixtureof nucleic acids, preferably comprising a segment of randomizedsequence, the SELEX method includes steps of contacting the mixture withthe target under conditions favorable for binding, partitioning unboundnucleic acids from those nucleic acids which have bound specifically totarget molecules, dissociating the nucleic acid-target complexes,amplifying the nucleic acids dissociated from the nucleic acid-targetcomplexes to yield a ligand-enriched mixture of nucleic acids, thenreiterating the steps of binding, partitioning, dissociating andamplifying through as many cycles as desired to yield highly specifichigh affinity nucleic acid ligands to the target molecule.

SOMAmers (Slow Off-rate Modified Aptamers) are aptamers having improvedoff-rate characteristics. This improved off-rate characteristic may berepresented as a rate of dissociation (t_(1/2)) or the point at which50% of the aptamer/target complex has dissociated. Such rates ofdissociation may vary, generally, from greater than 5, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220 and 240minutes, this being the average time it takes a protein-aptamer complexto dissociate. In addition, SOMAmers contain modified nucleosides thatprovide for different built-in functionalities. These functionalitiesmay include tags for immobilization, labels for detection, means topromote or control separation, hydrophobic sidechains to provide betteraffinity with proteins, etc. The modifications to improve affinity withproteins are chemical groups that are attached to the 5-position of thepyrimidine bases. By functionalizing the 5-position (e.g with a benzyl,napthyl or indole group) the chemical diversity of the SOMAmers isexpanded, allowing high affinity binding with a wider range of targetmolecules.

Additionally, some polymerases are still able to transcribe DNA withmodifications in these positions, thus allowing the amplificationnecessary for the SELEX process. SOMAmers, and the methods to producethem, are described in U.S. Pat. Nos. 7,964,356 and 7,947,447, bothentitled “Method for generating aptamers with improved off-rates,” eachof which is hereby incorporated by reference into the presentdisclosure.

It should be noted that while aptamers and SOMAmers may be discovered bythe SELEX process there may be other means to select them. For example,as computer modeling of molecular interactions improve, it may becomepossible to directly calculate an ideal nucleic acid sequence for anaptamer and the associated chemical modifications for a SOMAmer togenerate capture reagents specific to a given target molecule. Otherchemical techniques for screening for aptamers and SOMAmers besidesSELEX are also possible.

Assays directed to the detection and quantification of physiologicallysignificant molecules in biological samples and other samples areimportant tools in scientific research and in the health care field. Oneclass of such assays involves the use of a microarray that includes oneor more aptamers immobilized on a solid support. The aptamers are eachcapable of binding to a target molecule in a highly specific manner andwith very high affinity. See, e.g., U.S. Pat. No. 5,475,096 entitled“Nucleic Acid Ligands” see also, e.g., U.S. Pat. Nos. 6,242,246,6,458,543, and 6,503,715, each of which is entitled “Nucleic Acid LigandDiagnostic Biochip”. These patents are hereby incorporated by referenceinto the present disclosure. Once the microarray is contacted with asample, the aptamers bind to their respective target molecules presentin the sample and thereby enable a determination of the absence,presence, amount, and/or concentration of the target molecules in thesample.

Label-free assays are considered to be highly desirable, but are notalways achievable. A label is any foreign molecule that is chemically ortemporarily attached to the molecule of interest to detect molecularpresence or activity. Label-free assays utilize molecular biophysicalproperties such as molecular weight, molecular charge, dielectricconstant, or (in the case of the present invention) affinity for anaptamer to monitor molecular presence or activity. Some embodiments ofthe invention utilize a spin label linked to an aptamer attached to asurface. Because this spin label is attached to a component of thedetection system (e.g., an aptamer) as opposed to the molecule ofinterest (e.g., a protein), the present invention is considered to be alabel-free assay. Many sensitive assays require that the analyte be“labeled” with a detectable tag. This tag, or label, could be a dye, aradio-isotope, or anything else that is easily measured, therebymeasuring the analyte by proxy.

The ELISA assay (Enzyme-Linked ImmunoSorbent Assay) has been consideredthe “gold standard” of immunoassays due to its high sensitivity andspecificity, but is not considered to be label free. ELISA uses twoantibodies specific for different binding points (epitopes) on theanalyte, making it a “sandwich assay” as opposed to label-free. Oneantibody is immobilized to a surface, and captures the analyte from thesample fluid. The second antibody is linked to an enzyme that catalyzesa detectable change in a specific additive. After the analyte iscaptured by the immobilized antibody, the surface is washed to removenon-desired molecules, and the second antibody is added, followed by awash, and addition of the additive, which is usually catalyzed into adetectable dye. Although the second antibody adds both specificity anddetectability, it also requires several steps involving costly reagents.A label-free form of the assay would be comprised of only the firstantibody, and some means of detecting the binding of the analyte.

SUMMARY

Biochips, including protein chips, require a means of detecting theanalytes in a sample for which they are specific, while disregardingother molecules. The detection apparatus and methods described by thepresent teachings are based on color centers located close to thesurface of a solid that can be probed via Optically Detected MagneticResonance (ODMR) or other techniques. Color centers are point defects inotherwise close to ideal, transparent, crystalline insulators or largeband-gap semiconductors such as diamond, silicon carbide, or silica.They can consist of substitution defects where an atom in the crystal isreplaced by an atom of another type, vacancy defects where an atom ismissing, or combinations of the two.

Color centers have localized electron orbitals that are analogous tothose of a free atom. The electronic states are ordered in terms ofprincipal orbital and magnetic quantum numbers and can be stable whencharged, neutral, or both. The wide band-gap or insulating crystal thatsurrounds the color center plays the role of “vacuum” separating thecolor centers. At low enough density, this results in independent“atom-like” entities with a rich, well-resolved, complex energy spectrumwith discrete optical transitions in the visible range (hence the name“color center”) that co-exist with electronic and/or nuclear spin stateswith long relaxation times.

Of particular interest are color centers whose fluorescence intensitydepends on the spin polarization state (i.e. the magnetic quantum numberof the ground state). In this case the magnetic sublevel population isreflected in the fluorescence intensity, allowing for optical detectionof magnetic resonance. Because ODMR essentially transforms what would bedetection of radio frequency (RF) or microwave frequency quanta due totransitions in the magnetic sublevels into detection of a much higherenergy optical photon, it has a distinct advantage in sensitivity (about5 orders of magnitude) and for strong fluorescence, allows opticalobservation of individual color centers.

An example of such a color center capable of ODMR is thenitrogen-vacancy (NV) center in diamond crystals. Although the preferredembodiment described in the present invention would utilize NV centersin diamond for detection, the invention can also use other colorcenters. Diamond itself has over 500 known color centers, mostassociated with nitrogen. Other elements known as possible substitutionsin the diamond lattice include nickel, boron, silicon, hydrogen, andcobalt. Color centers in other crystalline lattices include, forexample, Germanium-related defects in germanosilicate glass, Siliconvacancies in silicon carbide, and X-ray induced defects in LiBaF₃crystals.

As the name implies, the NV center consists of a nitrogen substitutionfor a carbon atom situated next to a neighboring vacancy in the lattice.This is shown in FIG. 1 , which is a schematic depiction of a diamondcrystal, generally indicated at 100, including an NV center indicated at104. The NV center is a paramagnetic color center with unique couplingbetween its electronic spin states and optical states. It is capable ofemitting intense and stable fluorescence (i.e. large absorptioncoefficient combined with short lifetime of the excited state) and alsoexhibits very long magnetic relaxation times, making it a sensitivedetector of local properties such as the magnetic or electric fields.Diamond itself is exceedingly stable mechanically, thermally, andchemically. At the same time, the diamond surface is amenable tochemical modification, which is useful for attaching molecular agents.Pure diamonds are optically clear, allowing unfettered excitation andemission of fluorescent centers, and the fluorescence also exhibits nophotobleaching and minimal luminescent intermittency (“blinking”). Thecombination of all these qualities make NV centers a good candidate forvery sensitive biosensing.

The present disclosure describes devices, systems and methods fordetecting target molecules in a sample, based on a change in a propertyof one or more color sensors disposed near the surface of a substrate,when a target molecule binds to a capture reagent attached to thesurface. A detector can be configured to detect the change in the colorcenter property, thereby detecting the target molecule in the sample. Insome cases, the target molecule is a protein, and the capture reagentsare aptamers. In some cases, the color centers are NV centers disposedin diamond, and the change in property is a change in fluorescenceemission. In some cases, a large number of protein species may betargeted in one assay, such as hundreds, thousands, tens of thousands oreven more.

The present teachings involve the attachment of aptamers or SOMAmers ona surface, such as a diamond surface, as well as passivation of theregions of the surface not bound with aptamers or SOMAmers againstnon-specific binding by undesired proteins. The present teachings alsoinvolve regeneration of aptamers or SOMAmers attached to a surface forfurther rounds of protein detection.

The present teachings aim to eliminate all moving parts aside from thoserelated to bulk delivery of fluids to the active surface of the biochip.These fluids include the sample fluid, wash fluids, and fluids used forregeneration of the biochip. The proposed scheme for detection is alabel-free method, meaning that no detection agents need to be added tothe sample to enable the measurement of the proteins of interest.Regeneration of the active surface is non-destructive, requiring only amild buffer wash to dissociate the protein from the aptamer or SOMAmer.NV centers are extremely stable and aptamers and SOMAmers themselves arehighly stable, allowing for hundreds of uses.

More specifically, once the sample fluid has contacted the activesurface, some amount of time must be allowed for the proteins to bindthe immobilized aptamers. This binding time should also include timenecessary for the proteins to diffuse from the bulk fluid to thesurface, which may take hours. During this binding time, the samplefluid may be static, but it is more common to agitate or recirculate thefluid in order to bring unbound proteins closer to the surface andthereby reduce the distance they need to diffuse. After the binding stephas been completed, it is common to wash the surface to remove unboundproteins. This wash is commonly performed with buffer solutions similarto the sample fluid, such as phosphate buffered saline, or withdistilled water. When high specificity detection is required, the washesmay be harsher, using higher salt concentrations, mild denaturants suchas low concentration (<1 M) urea, or including competitors to theprotein of interest. Such competitors may be general, such as albumin,which competes with protein, or heparin, which competes with nucleicacids. Various bulk forms of nucleic acid can be used as well, such assalmon-sperm DNA, or bulk synthesized random DNA. The competitors mayalso be specific, such as the use of similar proteins to the protein ofinterest, or non-human proteins to compete with human proteins. As withbinding, the wash fluid may be static or agitated, and some time must beallowed for diffusion from the surface into the bulk fluid.

The present teachings eliminate most variance in sample analysis byperforming the assay and analysis at the point of collection, forexample, inside the test subject's toilet. Assaying and analyzing freshurine within the collection receptacle (namely, the toilet) eliminatesthe need to store and transport the samples, along with the associatedvariance.

The ability to perform routine proteomics cheaply at the level of theconsumer would have profound effects on science and healthcare.Proteomic science lags far behind its potential due to the simple lackof the large number of quality samples necessary for meaningfulanalysis. The present teachings greatly ease both the collection and theanalysis of such samples. Better proteomic science leads to bettermedical diagnostic predictions. However, such diagnostic predictions areof little utility in healthcare without ease of sample collection frompatients and ease of analysis. Thus, having provided for betterproteomic science, the present teachings will also provide for easierapplication of that science in healthcare.

Various other features of systems and methods according to the presentteachings are described in this disclosure. Features, functions, andadvantages may be achieved independently in various embodiments of thepresent disclosure, or may be combined in yet other embodiments, furtherdetails of which can be seen with reference to the following descriptionand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting a nitrogen vacancy center indiamond.

FIG. 2 is a schematic depiction of target molecule detection, accordingto aspects of the present teachings.

FIG. 3A is a schematic depiction of an interaction mechanism that may beused to detect target molecules, according to aspects of the presentteachings.

FIG. 3B is a schematic depiction of another interaction mechanism thatmay be used to detect target molecules, according to aspects of thepresent teachings.

FIG. 4 is a schematic depiction of electron energy levels in a diamondnitrogen vacancy (NV) center, and the transitions between these levels,according to aspects of the present teachings.

FIG. 5 depicts exemplary optical absorption and emission spectra of anNV center, according to aspects of the present teachings.

FIG. 6 is a schematic representation of reading and spin polarizing anNV center optically, according to aspects of the present teachings.

FIG. 7A is a schematic representation of excitation and emission pulsesassociated with an NV center, according to aspects of the presentteachings.

FIG. 7B is a graphical representation of NV center emission intensity,comparing the intensity as a function of time in the absence andpresence of a bound target molecule, according to aspects of the presentteachings.

FIG. 8A depicts a first illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings.

FIG. 8B depicts another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings.

FIG. 8C depicts yet another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings.

FIG. 8D depicts still another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings.

FIG. 9 depicts still another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings.

FIG. 10 is a flow chart depicting illustrative steps in a method ofdetecting target molecules, according to aspects of the presentteachings.

FIG. 11 is a flow chart depicting illustrative steps in another methodof detecting target molecules, according to aspects of the presentteachings.

FIG. 12 is a flow chart depicting illustrative steps in yet anothermethod of detecting target molecules, according to aspects of thepresent teachings.

FIG. 13 is a flow chart depicting illustrative steps in a method ofmanufacturing a device for detecting target molecules, according toaspects of the present teachings.

DESCRIPTION

FIG. 1 is a schematic diagram depicting a nitrogen vacancy center indiamond. In its pure form, a diamond crystal 100 would consist solely ofcarbon atoms 101. When a nitrogen atom 102 substitutes for a carbon atomin the diamond crystal, and is located adjacent to a vacancy 103 in thecrystal, a nitrogen-vacancy center 104 is created.

As can be seen in FIG. 1 , since each carbon has four identical bondingpartners, there are four crystallographic directions upon which the axisof the NV center can lie depending on how the vacancy is placed relativeto the nitrogen substitution. These are the [111], [11 1], [111], [1 11]directions. Additionally, in any given direction, the order of thenitrogen substitution and vacancy can be reversed so there are, in fact,eight unique NV center configurations.

The interactions of a molecule with the NV center depend not only on thedistance to the NV center but also on the relative orientation of thetwo. Additionally, there are optimal directions for the excitation anddetected light. All of these considerations must be taken into accountwhen choosing which face of the diamond crystal should be used as thedetecting surface.

Nitrogen-vacancy centers may be embedded in a crystalline structure,such as a diamond, at the desired depth by introducing the nitrogenimpurities and the vacancies at the desired depth, then annealing from1000K-1300K, which allows the vacancies to collocate via diffusion tothe nitrogen impurities. Nitrogen defects may be implanted at thedesired depth either through a nitrogen pulse during chemical vapordeposition (CVD) of the diamond matrix or by ion beam implantation afterthe deposition has completed. Vacancies are implanted or created via ionbeams of e− [54], H+ [55] or He+.

Because natural diamond contains about 1% ¹³C, which has a ½ nuclearspin (it is a nuclear paramagnet) and therefore interacts with NVcenters, the relaxation times of NV centers can be increased by growingan overlayer of isotopically pure ¹²C diamond on an existing crystalusing CVD and creating NV centers within this layer. Similarly, the ½nuclear spin of ¹⁵N is preferred over the nuclear spin 1 of the ¹⁴Nisotope. Also, as the nitrogen substitutions carry a spin, the largerthe percentage of nitrogens that can be converted to NV centers thecleaner the sample will be from a magnetic perspective.

Under the proper CVD conditions, the orientation of NV centers createdcan be highly biased. That is, rather than all 8 possible orientationsbeing equally populated, a single orientation can occur preferentiallyover the others. Alignments as high as 99% may be possible. This can bevery useful for creating a device where all of the NV centers areidentical and optimally oriented. For example, if the sensing surface ofthe diamond is a (111) plane, and the great majority of NV centers canbe oriented along the [111] direction, they will all be orientedperpendicular to the sensing surface.

For some embodiments of the present teachings, it is desirable to createa very thin layer of diamond attached to another substrate, such assilicon, where additional detection electronics exist. Another use forion implantation is to insert a “break layer” at a certain depth in athick diamond crystal using, for example, hydrogen atoms. After bondingthe thick diamond crystal to the other substrate, the diamond can bemechanically shocked and will fracture along the break layer leavingbehind a thin layer of diamond crystal. Similar techniques are used inthe semiconductor industry already.

FIG. 2 is a schematic depiction of target molecule detection, accordingto aspects of the present teachings. A capture reagent 200 is attachedto the surface 202 of a substrate 203 (e.g., a high-purity insulatorsuch as a crystalline film, a diamond film, and/or a single-crystaldiamond) in close proximity to a color center 204. When the color centeris irradiated with excitation light 208 (e.g., from optical source 209),it is stimulated to emit fluorescent light 210, characterized by aspectrum and intensity. Capture reagent 200 is contacted with a samplefluid 211 (e.g., by exposing surface 202 to the sample fluid) such thata target analyte 206 (also called a target molecule) in the samplesolution binds to the capture reagent 200 to form a complex 212, therebycausing and/or changing an interaction 214 with the color center, whichproduces a detectable change in properties (e.g., intensity) of theemitted fluorescent light 216. In interaction 214, the color center maydetect electric field changes or magnetic field changes upon binding ofa target molecule to a capture reagent. A property of the color center(e.g., a property associated with a magnetic resonance or spin of thecolor center) be changed by interaction 214.

The sample fluid may be a biological fluid. Capture reagent 200 may be anucleic acid molecule, an oligonucleotide, an aptamer, a SOMAmer, or anyother binding agent configured to bind to a target molecule. In someexamples, the capture reagent includes at least one 5-position-modifiedpyrimidine (i.e., a pyrimidine, such as a uridine or a cytidine, havinga modified 5-position). In some examples, different types of5-position-modified pyrimidines are attached to the surface. Forexample, at least one 5-position-modified uridine and at least one5-position-modified cytidine may be attached to the surface.

The target analytes may be a protein. Proteins include normally foldedpolypeptide chains, abnormally folded polypeptide chains, unfoldedpolypeptide chains, fragments of a polypeptide chain that may or may notbe normally folded, short polypeptides, polypeptides that incorporatenon-natural amino acids, and polypeptides that are post-translationallymodified (e.g., phosphorylation, glycosylation, disulphide bonding,etc.) or polypeptides assembled into a protein complex. The targetanalytes may also include small molecules found in biological fluidssuch as metabolites.

FIGS. 3A-3B are schematic depictions of two possible interactionmechanisms of the detection technique where the color centers are NVcenters, the target analytes are proteins, and the capture reagents areaptamers.

FIG. 3A is a schematic depiction of an interaction mechanism that may beused to detect target molecules utilizing magnetic spin labels,according to aspects of the present teachings. Stable organic moleculesare typically diamagnetic, the paramagnetic molecules (i.e. “freeradicals”) possess unpaired electrons, therefore they are chemicallyactive, and prone to lose their spin through chemical reactions. Stablefree radicals that survive exposure to a bio-chemical environment areexceptional. Spin-labels are stable paramagnetic organic molecules orcomplexes that are capable of binding to another molecule, particularlyto nucleic acids and amino acids, specifically developed forsite-directed spin-labeling of large bio-molecules.

The spin labels most often used are nitroxide-containing small organicmolecules, or metal chelators such as EDTA that complex with highaffinity to paramagnetic metal ions, developed for incorporation intonucleic acids. Nitroxide derivatives of nucleic acid bases have alsobeen developed. Recently, triarylmethyl (Trityl) radical derivativeshave also become popular for labeling both proteins and nucleic acids.EDTA derivatives of both deoxyribo-thymine and deoxyribo-cytosine havebeen developed in phosphoramidite form for use in DNA synthesizers. Formeasurements where the spin label is intended to simply change therelaxation time of NV center, all that is important is that a magneticinteraction exists and a wide variety of spin labels are applicable.However, for measurements where the spin states of the label aredirectly addressed, the individual spectra of the spin label becomesrelevant. For example, in DEER, the narrower the spectrum and the longerthe lifetime of the spin label, the better. As a specific example, inDEER the triplet splitting of the spectrum due to hyperfine coupling tothe ¹⁴N is a handicap, and the single line spectrum of deuterated Tritylis a preferred choice.

In FIG. 3A, an aptamer 301 linked to a magnetic spin label 300 isattached to the sensing surface 202 of a diamond crystal 100, in closeproximity to a color center 104. A target protein 302 in the samplesolution binds to the aptamer to form a complex 303, changing theproximal relationship of the spin label with the NV center which affectsthe interaction 304 between the spin label and the NV center, therebyproducing a detectable change in fluorescent properties 306 of the NVcenter.

More specifically, FIG. 3A illustrates a specific configuration designedto maximize the magnetic interaction by placing a spin-labeled aptamerin the proximity of an NV center in diamond. The conformational changeupon binding of the protein can move the spin label relative to the NVcenter. A movement closer to the NV center will increase the magneticfield at the NV center resulting in a measurable change in the dynamicor quasi-static characteristics of the NV center sublevel spectrum.

The sublevel spectrum may be probed using a detector assembly configuredto irradiate the NV center with excitation light and to detect emissionof electromagnetic radiation from the NV center. The detector assemblymay detect changes in the sublevel spectrum by irradiating the NV centerwith microwave radiation over a range of frequencies including aresonant frequency of the sublevels, and detecting changes in theradiation emitted by the NV center. Radiation at the resonant frequencymay induce conversion of ground-state electrons in the NV center from afirst sublevel to a second sublevel and thereby induce a change in acharacteristic of the emitted radiation. An example characteristic ofthe emitted radiation is the relationship between the frequency of theexcitation light and the frequency of the emitted light. Furthermore,the addition of a spin label along with microwave excitation open avariety of options to manipulate spin label interaction with the NVcenter.

FIG. 3B is a schematic depiction of another interaction mechanism thatmay be used to detect target molecules utilizing electrostaticinteractions, according to aspects of the present teachings. An aptamer301 is attached to the sensing surface 202 of a diamond crystal 100 inclose proximity to an NV center 104. Both positive ions 308 and negativeions 309 are present in the fluid being analyzed. The aptamer'snegatively charged backbone will attract positive ions 308. Theseassociated ions affect the electric field local to the NV center, sothat when the NV center is irradiated with excitation light 208, it isstimulated to emit fluorescent light 310, characterized by a spectrumand intensity. A protein 302 in the sample solution, also has ionsassociated with it according to its charge. The protein binds to theaptamer to form a complex 303, thereby changing the distribution ofcharge, and thus the electric field local to the NV center, therebyproducing a detectable change in properties of the emitted fluorescentlight 312.

More specifically, FIG. 3B illustrates a configuration whereelectrostatic interactions can be explored. The presence of mobilecharges near the surface significantly affects the relaxation time of NVcenters. There is a negative charge associated with the NV center itselfand also negative charges associated with the backbone of the aptamer.Additionally, the target proteins themselves can be charged. Thesecharges will be screened by counter-ions in the solution. Binding of aprotein molecule to an aptamer will rearrange this charge distributionrelative to the NV center. Furthermore, the protein molecule itself hasa dielectric constant much lower than water and its presence will affectthe screening of the charge distribution. Both effects will give rise toa change in the quasi-static and dynamic electric fields present at theNV center and affect the characteristics of the sublevel spectrum.

The degree to which static or dynamic changes in the sublevel spectrumdominate the interaction can be affected by the environment in which themeasurement is made. If the measurements are made at room temperature,the mobility of charges in solution and the molecules themselves areexpected to contribute significant dynamic changes that can becharacterized by direct measurement of T₁. In contrast, if themeasurements are made at a temperature below freezing of the biologicalfluid, these dynamic changes should be suppressed and allow directdetection of the static splitting in the sublevels. In the case of thespin-labeled aptamer, making the measurement in a frozen state willsignificantly increase the relaxation time of the spin label and allowtechniques like double electron-electron resonance (DEER) to be used tomeasure directly the distance change between the spin label and the NVcenter.

The optimum configuration for the placement of the NV centers alsodepends on whether the changes in the spectrum are dominated byquasi-static or dynamic changes. If dynamic changes are dominant anddirect T₁ measurements are being used, it is likely that the NV centerscan be located closer to the surface and also closer to each other. Onthe other hand, careful measurements of quasi-static changes to thespectrum will require the high fidelity of longer relaxation times(primarily T₁) and hence require NV centers further from the surface andwell separated from each other. The NV center distance from the surfaceis a compromise between maximizing the interaction with the aptamer andits binding target and preserving the long relaxation times that makethe NV sensitive detectors of quasi-static electric and magnetic fields.

FIGS. 3A-3B explored two limits of interaction where the magnetic andelectric fields are the dominant ones causing a change in the NV centerupon binding of the analyte. Just as the SELEX process was modified toscreen for slow off-rates in the creation of SOMAmers, a step can beadded to later selection rounds to bind candidate aptamers to a devicelike those described here and measure NV center response to targetbinding. In this way, SOMAmers that maximize the signal contrast upon abinding event can be maximized.

FIG. 4 is a schematic depiction of electron energy levels in a diamondnitrogen vacancy (NV) center, and the transitions between these levels,according to aspects of the present teachings. More specifically, FIG. 4is a simplified schematic of the electron energy levels in a negativelycharged NV center in diamond and the transitions between these levels. Aground state 399 of an electron in an NV center includes three sublevels(also called substrates or states). The zero-spin sublevel 400 has thelowest energy, and is designated |³A₂,0>. The m_(s)=+1 and m_(s)=−1 spinsublevels 402 have identical energy in the absence of external fields,and are designated |³A₂,±1>.

External fields affect the m_(s)=±1 spin states but not the m_(s)=0 spinstates, lowering the energy of the m_(s)=−1 spin states while raisingthe energy of the m_(s)=+1 spin states. For instance, a 1027 Gaussmagnetic field that is aligned with the symmetry axis of the NV centerwill lower the |³A₂,−1> level to that of the |³A₂,0> level at roomtemperature. The presence of a magnetic spin label in close proximity tothe NV centers would have a similar, but much smaller effect.Irradiation with microwaves of the correct frequency 404 (e.g., aresonant frequency) will induce transitions from the ground m_(s)=0 spinstate to the ground m_(s)=±1 spin states. Transition from the groundm_(s)=0 spin state to the excited m_(s)=0 spin state 406, designated|³E,0>, or from the ground m_(s)=±1 spin states to the excited m_(s)=±1spin states 408, designated |³E, ±1>, may be induced by irradiation withexcitation light of the correct frequency 208.

Transitions from the excited states to their corresponding ground statesare accompanied by emission of red photons 418. Alternatively,transitions from the excited states to the ground states may occur viathe “dark transition” 410, so called because it most or all of thetransition is accomplished without any photon emission or only withlonger wavelength infrared photon emission 412. The dark transition 410may either be accompanied by the emission of a photon 412 in theinfrared range which will be absorbed by the diamond lattice, or along apath 414 with no photon emission at all. In most cases, the darktransition results in a spin transition 416 from |³E, ±1> to |³A₂,0>.Approximately 30% of the electrons on the |³E, ±1> level transition tothe ground state via the dark transition, producing no detectable photonemission. Because of this, electrons in the m_(s)=±1 spin states produceonly about 70% of the fluorescence of electrons in the m_(s)=0 spinstate.

Even more specifically, FIG. 4 shows a schematic energy level structureconsistent with the observations and calculations for a negativelycharged NV center. Six electrons are located in a C_(3V) symmetry(imposed by the static lattice structure) ground state “molecularorbital”. These orbitals are linear combinations of the four orbitalsconsistent with the band structure calculation of diamond.Energetically, this ground state, labeled as ³A₂ in FIG. 3 , is a deeplevel state in the band gap. The first excited state, labeled as ³E, is1.94 eV above the ground state. While it is less localized, it is stillmore than 1 eV from the conduction band. The 1.94 eV difference betweenthe two bound states corresponds to the observed 638 nm zero phononabsorption and emission line shown on FIG. 4 .

While both the ground state and the excited state are S=1 spin triplets,going forward we concentrate on the magnetic sublevels of the groundstate only. Thus rather than using the cumbersome state notation in FIG.3 , we will often simply refer to m_(s)=0, or m_(s)=±1 states, it beingunderstood we are referring to the ground state.

In the absence of applied fields, the degeneracy of the m_(s)=0 andm_(s)=±1 sublevels in the ground state is lifted first by a large zerofield splitting (2.88 GHz) along the NV center axis, reflecting thestrongly non-spherical wave function of the 6 electrons. In the presenceof applied magnetic and/or electric fields, the m_(s)=+1 and m_(s)=−1sublevels are further split. For fields parallel to the NV center axis,the splitting is 2.8 MHz per gauss for a magnetic field, and 3.5 mHz perV/m for an electric field. Note that the figure does not representenergy differences to scale, the magnetic sublevels splitting beingabout 5 orders of magnitude smaller than the principal split (1.95 eV)between the ground state and the excited state.

Transitions between the ground-state sublevels 400 and 402, and theexcited states 406 and 408 lie in the visible range and give rise to theinteresting photoluminescent properties of the NV center. FIG. 5 depictsexemplary optical absorption and emission spectra of an NV center atroom temperature, according to aspects of the present teachings. Theabsorption spectrum 500 peaks around 570 nm, and shows how efficientlyphotons of given wavelengths are absorbed by the NV center. The emissionspectrum 502 peaks around 690 nm, and shows the relative intensity oflight emitted at given wavelengths. The absorption of photons of anywavelength in the absorption spectrum can lead to emission of photons ofany wavelength in the emission spectrum. The spreading of the spectrafrom the expected discrete wavelengths is due to dissipation ofvibrational energy into the molecular lattice, or absorption of thermalenergy from the molecular lattice. Both spectra share a peak 504 wherethe energies of the absorbed and emitted photons are identical. Thispeak is called the zero phonon line, and is where these spectra wouldconverge to as the ambient temperature approaches absolute zero andlattice vibrations are frozen out.

More specifically, both the absorption and emission spectra show a vastphonon broadening resulting in a strong absorption coefficient, a short(˜4 ns) lifetime for the excited state and a practically non-bleaching,intense fluorescence. The direct absorption and emission paths leaveboth S and m_(s) unchanged (the angular momentum of the absorbed oremitted photon is compensated by a “molecular” orbital momentum change).In contrast, the strong phonon coupling also allows for alternativedecay path(s) (via inter-system crossings), which change m_(s) by one,as shown on right side of FIG. 3 . This decay path goes through multipleintermediate states, and any photon energy emitted are much further intothe infrared compared to photons generated by the direct path. Hencethis is often called a “dark path”, and is taken with a higherprobability for electrons excited from the m_(s)=±1 sublevel than thoseexcited from the from the m_(s)=0 sublevel. The net result is a highervisible fluorescence intensity for electrons excited from the m_(s)=0sublevel of the ground state compared to those excited from the m_(s)=±1sublevels.

FIG. 6 is a schematic representation of reading and spin polarizing anNV center optically, according to aspects of the present teachings. Anexcitation pulse 600 irradiates a nitrogen-vacancy center, stimulatingan emission pulse 602. The initial intensity 604 of the emission pulse,I_(start), is the lowest point of the pulse and is indicative of therelative population of the m_(s)=0 and m_(s)=±1 states at the beginningof the measurement. The intensity rises rapidly to the maximum intensity606, I_(max), as the spin states become polarized to the m_(s)=0 state.

Changes in the magnetic sublevel populations are exactly what areobserved via Electron Paramagnetic Resonance, EPR, where the frequenciesof the applied microwave excitations match the splittings of the m_(s)sublevels. The fluorescence intensity depends on the sublevelpopulations as described above, allowing Optical Detection of the groundstates's Magnetic Resonance (ODMR) on NV centers. Note that while wewill continue to refer to ODMR detection, direct electrical detection ofphotocurrents in NV centers have been demonstrated and could serve as analternative detection mechanism.

Traditional magnetic resonance applies continuous or pulsed microwave/RFelectromagnetic fields to induce transitions between or to mix themagnetic sublevels of large (>10¹³) numbers of spins at the same time,i.e. operating on the macroscopic (ensemble average) magnetization,which depends on the temperature. At zero absolute temperature all spinsare in their lowest energy sublevel and the system magnetization ismaximum, therefore this state is 100% spin polarized. At finitetemperature the sublevel population follows Boltzmann statistics,decreasing the ensemble-averaged polarization with increasingtemperature. At room temperature—as the energy difference between thesublevels is very small—the population of the different magneticsublevels is almost equal, resulting in only a 0.1% polarization.

Magnetization originating from the population difference of thesublevels is referred to as the longitudinal magnetization, or the zcomponent of the magnetization as conventionally the z-axis is chosenparallel with the quantization axis. At any temperature, thelongitudinal magnetization has an equilibrium value which isproportional to the spin polarization. The transverse magnetization, theXY component of the magnetization, is the ensemble-average of mixedstates (as the eigenstates lie along the z direction). Its equilibriumvalue is zero.

Sublevel transitions or mixed states (with temporarily non-zero ensembleaverage of the transverse magnetization) can be created by applyingRF/microwave electromagnetic fields with photon energies matching theenergies of the sublevels splitting E. The corresponding frequency isdefined by E=hf_(L), where h is the Planck constant and f_(L) is knownas the Larmour frequency. The mixed states (and hence the ensembleaverage transverse magnetization) precess (rotate) at the Larmourfrequency. Typically, the EMF induced by this rotating macroscopicmagnetization is detected through a coil or resonator.

After the creation of mixed states, the transverse magnetization signaldecays in time principally for two reasons:

-   -   1. The individual spins experience different fields or changing        fields during the precession, i.e. the energy difference of the        m_(s) sublevels are different, therefore the precession        frequency of the mixed states are slightly different. While they        begin precessing in phase, phase differences will build up due        to the differences in the precession frequencies and the        ensemble average (vector sum) transverse magnetization will go        to zero. As the precessing macroscopic magnetic moment decays to        zero, naturally the induced signal decays as well. The        characteristic time of this decay, caused by the loss of “phase        coherence” is referred to as T₂ or the transverse relaxation        time. In more elaborate experiments, decay due to static field        differences (i.e. magnetic field inhomogeneity over the sample)        is eliminated and the decay time, reflecting only dynamical        changes during the measurement, is then referred to as phase        memory time T_(M). Loss of the transverse magnetization in this        fashion does not require energy exchange with the environment.    -   2. At finite temperatures, spins can exchange energy with the        environment and flip from one sublevel to another, i.e. the        eigenstates have a finite lifetime due to interactions with the        environment. More precisely, the fluctuating (time dependent)        electromagnetic field generated by the environment has to have a        non-zero Larmour frequency field component in the transverse        direction to induce a state change. As a result of these        changes, the precessing transverse magnetization will be lost as        the magnetization turns back to the z (or longitudinal)        direction. The characteristic time associated with this process        (the time required to return to thermal equilibrium population        distribution of eigenstates) is called T₁, or longitudinal        relaxation, or spin-lattice relaxation time, as it requires        energy exchange with the environment. This process imposes an        upper limit on any precession decay times, such as T₂ and T_(M).        Unlike traditional EPR detection, ODMR detects the longitudinal        magnetization. The loss of transverse detection and its        advantages, however are abundantly compensated by a huge        sensitivity gain: low energy magnetic sublevel transitions can        be observed via optical photons with 100,000 times greater        energy, at near perfect quantum efficiency.

Besides allowing for ODMR detection, in isolated NV centers the m_(s)dependent fluorescence also allows manipulation of the sublevelpopulations. This is because the lifetime of the magnetic sublevels aremuch longer than the fluorescence lifetime. At high enough excitationintensities, the fluorescence rate is limited only by the fluorescencelifetime and can be 100 MHz or higher. For example, if an excitationfrom the m_(s)=±1 sublevel of the ground state has a w=0.7 probabilityof retaining its quantum number and a 0.3 probability of returning tothe m_(s)=0 sublevel via the dark path, then after 10 cycles theprobability to find any of the spins in the m_(s)=0 state will be 98%,i.e. a 98% spin polarization can be built up for a macroscopic sample.As T₁ of NV centers is typically much longer than 100 microseconds, asufficiently intense 100 ns pulse can create a spin polarizationequivalent to cooling the diamond from room temperature to 0.3K.Starting any magnetic resonance measurement with a nearly fully spinpolarized state is a 1000× gain in signal compared to starting with theroom temperature equilibrium spin polarization.

The same pulse that polarizes the spin population also acts as ameasurement of the spin polarization, as shown in FIG. 6 . The maximumfluorescence arises when the sample is 100% spin polarized in them_(s)=0 sublevel. We define that maximum fluorescence intensity asI_(max). If we use the probabilities from the above example, assuming weare starting in thermal equilibrium, then when the excitation light isturned on at t=0 the fluorescence response will be 80% of I_(max)corresponding to ⅓ of the population in the m_(s)=0 sublevel fluorescingat max efficiency and ⅔ in the m_(s)=±1 states fluorescing at 70%efficiency (⅓+⅔*0.7=0.8). The intensity will asymptotically approachI_(max) at a rate determined by the intensity of the excitation light.

In this case, the initial polarization of the m_(s)=0 sublevel, P₀, canbe expressed in terms of the initial fluorescence intensity I_(start),as

$P_{0} = {\frac{1}{\left( {1 - w} \right)}\left( {\frac{I_{start}}{I_{\max}} - w} \right)}$In the case where w=0.7 analyzed above, if I_(start)=I_(max), then P₀=1.If I_(start)=0.7*I_(max) then P₀=0. If I_(start)=0.8*I_(max), then P₀=⅓,etc.

The unique properties of the NV center outlined above means that it iseasy to construct an experimental setup which is able to detect thefluorescence of a single NV center using a commercial fluorescencemicroscope. As the NV centers can be separated by hundreds ofnanometers, the fluorescence of single NV centers observed with themicroscope carries all the spectroscopic information of the magneticsublevels of a single spin. As a single spin is being observed there isno ensemble average as is the case in traditional EPR measurements, butthere is still a direct correspondence between ensemble average and thetime average of the single spin. To relate the two, polarization valuesof the ensemble average have to be replaced with probabilities ofsublevels, i.e. an optical excitation pulse which leads to 98% m_(s)=0spin polarization of a macroscopic ensemble will translate to findingthe single NV center in an m_(s)=0 state with 98% probability. Thanks tothe high fluorescence, fast repetition and efficient time averaging,reasonably short experiment times with small statistical errors can beachieved.

The properties of NV centers are well suited for time domain magneticresonance: the 100% polarized initial state, single center sensitivitydetection, and long relaxation times, plus the fact that all time-domainrf/microwave pulse sequences ever invented for sophisticated, highresolution spectroscopy can be applied, render a single NV center one ofthe most sensitive quantum measurement tools available.

FIGS. 7A-7B depict a means to measure the relaxation time, T₁, through apurely optical measurement. The m_(s)=±1 spin sublevels emit with afluorescent intensity approximately 70% of that of the m_(s)=0 sublevel,so that if equally partitioned, the total fluorescent intensity is about80% of what it would be if the spins are fully polarized into them_(s)=0 sublevel. A series of pulses, as in FIG. 6 , allow formeasurements to assess the relative populations of the m_(s)=0 andm_(s)=±1 sublevel and also repolarize the spins into the m_(s)=0sublevel.

FIG. 7A is a schematic representation of optical excitation pulses 708and emission pulses 710 associated with an NV center, according toaspects of the present teachings. An NV center in diamond is irradiatedwith a series of pulses of excitation light, each of long enoughduration to cause full polarization of the NV center into the m_(s)=0sublevel. The spacing τ_(i) between these excitation pulses is varied.The fluorescent intensity immediately after the start of the excitationpulse (filled squares I₀, I₁ . . . ) is measured, as is a referenceintensity (open rectangles), taken when the spin states should be fullypolarized in the m_(s)=0 sublevel.

FIG. 7B is a graphical representation of NV center emission intensity,comparing the intensity as a function of time in the absence andpresence of a bound target molecule, according to aspects of the presentteachings. More specifically, the difference between the initialintensities and the reference intensities in FIG. 7A is plotted as afunction of the time spacing τ_(i) between them and shows how long isrequired for the sublevels to return to thermal equilibrium. Anexponential fit of form Intensity=I₀*exp(−τ/T₁) yields a direct measureof T₁. The data points 700 show a faster return to equilibrium and thustheir exponential fit 702 would yield a shorter T₁ than the data points704 and their exponential fit 706. Different T₁ values arise as aconsequence of an analyte being bound or not bound to the aptamers.

More specifically, in FIGS. 7A-7B, a series of excitation pulses ofsufficient duration and intensity to optically polarize the NV centerare applied with variable waiting times between them. This excitationwavelength is shorter than the zero phonon line, and in practice 532 nmis often used. At the same time the excitation pulses are applied, theemission from the NV center is monitored in a bandwidth that includessome or all of the emission spectrum 502.

Initially, the ground state is thermally equilibrated, thus the initiallevel of fluorescence I₀ corresponds to a level less than the maximumpossible I_(m) because the m_(s)=±1 and m_(s)=0 states are almostequally populated. Over the course of the initial pulse, the emissiongrows to a maximum and saturates at I_(m) as the m_(s)=0 state becomespopulated due to the dynamic spin polarization process explained above.After a waiting time, a second pulse is applied. If the waiting time isshort enough, interactions with the surrounding environment will nothave had enough time to equilibrate the populations of the ground statesand the m_(s)=0 will still be highly populated and thus the initialemission intensity I₁ that is measured will still be close the maximumI_(m).

Spin polarization will again take place during the remainder of thesecond pulse and the state will again be polarized to m_(s)=0 and acorresponding intensity of I_(m). A longer waiting time then ensues, andwhole process is repeated again. As this process is repeated eventuallythe waiting time will become substantially longer than the relaxationtime of the NV center, and the ground state will be thermallyequilibrated and all three sublevels will be equally populated and theinitial emission measured will be equal to I₀ again.

By plotting the initial emission intensity I₁, I₂, I₃ . . . as afunction of the waiting time, a graph such as that in FIG. 7B can begenerated. A fit to the graph can yield the relaxation time T₁, of theNV center. FIG. 7B shows an example of two data sets and theirrespective fits corresponding to different T₁ values. Note that thismeasurement sequence is highly analogous to that used in a standardpulsed magnetic EPR experiment, where the initial state is the invertedthermal equilibrium magnetization −M₀, prepared by a microwave π pulseand the later time value is read out by detecting microwave radiationinitiated by another microwave π/2 pulse. However, in this case nomagnetic field or microwave antenna or resonator is required, thusconsiderably simplifying the measurement.

With the addition of microwaves, more detailed measurements techniquesbecome possible. For example, in the case of the purely optical T₁measurement explained above, a microwave π pulse (with the frequency ofthe zero-field splitting and with the field oriented perpendicular tothe NV center symmetry axis) added right after every polarizing lightpulse would provide an initial population of zero for the m_(s)=0 state.By alternating the measurement with and without microwave pulses andplotting and fitting their difference, one could measure T₁ without theneed of fitting for the value of the residual (thermal equilibrium)signal. Note, that this works so simply because in the absence of anexternal magnetic field the m_(s)=+/−1 states are degenerate (equalenergy).

Adding an external field further increases the possibilities formeasurement. When an external static field H₀ is imposed, the m_(s)=±1states energy levels split, so the m_(s)=0 to m_(s)=+1 and the m_(s)=0to m_(s)=−1 transitions will respond to different frequency microwaveexcitations. As long as the applied field is small or parallel with theNV center symmetry axis the optical response will not change and cancontinue to be used for readout. Optical polarization of the m_(s)=0state continues to serve as an ideal starting point for experiments andthe initial fluorescence intensity measured during an optical excitationpulse at a later time can be used to measure the population of them_(s)=0 sublevel, i.e. serve as sensitive longitudinal detection. Thetime interval between the initial polarization and the detection can bevaried and used to perform sublevel spectroscopy between levels selectedby the frequency of the microwave pulses. Note than an external electricfield will also split the sublevels and can be used rather than anexternal magnetic field.

All known microwave pulse sequences ranging from simple Hahn echos,Carr-Purcell-Meiboom-Gill (CPMG), to MREV-8 can be used to explore theNV centers coherence time, i.e. determine small field variations down toa few tenths of milliGauss over millisecond time scales. Theprerequisite for these measurements is a long T₁, requiring not only NVcenters further from the surface (15-20 nm) but also freezing the motionof the aptamers, proteins and ions. i.e. freezing the liquid studied.

Additionally, distance changes between a spin-label attached to theaptamer and the NV center induced by the protein capture can be measuredwith Double Electron—Electron Resonance (DEER) experiments. Applying asecond microwave excitation at a different frequency to reverse the spinon the spin-label while reversing the precession of the NV sublevels,the precession frequency difference due to the spin reversal can bedetected with high precision, enabling sub-nanometer precision distancemeasurements up to 20 nm. To perform DEER over the distances required,the relaxation time of both the NV center and the spin-label on theaptamer will need to be of the order of milliseconds, again requiringfreezing the liquid being studied.

As described above, the lateral separation between the NV centers may belarge, such as more than 100 nm, to prevent significant interactionsbetween NV centers. The aptamers, however, are small, of order a fewnanometers, so we distinguish two different realizations. In the first,aptamers are bound to the surface densely so that any given NV centerinteracts with multiple aptamer sites distributed randomly within the NVcenter's range of interaction and thus measures an average interactionchange across the multiple sites.

In a second possibility, site-directed surface chemistry is used toensure a one to one pairing between aptamer sites and underlying NVcenters. This can be achieved by using surface chemistry influenced bythe charge present in the NV center, or by the fluorescence of the NVcenter. This configuration has the advantage of producing nearlyidentical measurement sites affording single protein sensitivity and theability to discriminate non-specific binders from the correct targetproteins. This ability to assemble statistics on the basis of yes or nodecisions on a molecule-by-molecule basis means that given the samenumber of NV centers, the second configuration may provide superiorresolution and dynamic range.

One goal of the present teachings is to measure the concentration ofmultiple proteins simultaneously by using aptamers specific to eachindividual protein. There are at least two fundamentally different waysthat this can be accomplished.

In a first possible detection paradigm, for any given protein targetthere are a number of individual NV centers and aptamer sites that areeach measured by an independent detector. This is effectively running amultitude of single NV center experiments. If there is further aone-to-one pairing between NV centers and aptamer sites, then one cancount the number of bound proteins to generate a single numberindicative of the concentration for that specific target protein.

In the second possible detection paradigm, a multitude of NV centers andaptamer sites for a given protein target are measured simultaneously bya single detector. While this foregoes the molecule-by-moleculediscrimination advantages and will require creating calibration curvesto quantify the concentration of the target protein, it may reduce therequired complexity of the device.

In either case, ideally all NV centers are excited and detected at thesame time, however, that does not preclude using multiple measurementprotocols to address the fact that different aptamers may have differentresponses.

For both cases, different portions of the diamond (or more generally,crystalline) surface will be dedicated to measuring the concentration ofdifferent proteins. However, a difference arises in the number ofdetectors required. For example, detecting the concentration of 10,000proteins could require on the order of 10⁸ detectors in the first case,but would only require 10,000 detectors in the second case.

FIGS. 8A-8D depict different configurations for probing a biochip basedon the present teachings. While identical NV centers lying along the[111] direction close to a (111) terminated diamond surface would be anideal configuration because all NV centers being measured are equivalentand the most sensitive detection area is located directly above the NVcenter, other constraints, such as which orientation diamond crystalscan be easily manufactured, may not allow this. Some deviceconfigurations will be more forgiving of this situation than others.

FIG. 8A depicts a first illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings. In the simplest configuration aptamers 301 are attached tothe surface of a diamond crystal 100, in close proximity tonitrogen-vacancy centers 104 located at a prescribed depth below thesensing surface 202. Properties of the fluorescent emission light 418,which is induced or stimulated by excitation light 208, are measured inorder to detect the presence of protein-aptamer complexes 800. Thepresence of the protein induces a change in one or more properties ofthe nitrogen-vacancy center, and the change is detected based on thefluorescent emission from the nitrogen-vacancy center.

The configuration shown in FIG. 8A is all that is required to performthe all-optical T₁ measurement described above. It imposes the leaststringent conditions for the NV center equivalence. A (100) diamondsurface with all 8 different possible NV center orientations could beused. In this case the NV center symmetry axes won't be perpendicular tothe surface, which isn't ideal, but all 8 orientations will bemisaligned by the same amount so the NV centers perform equivalently.One disadvantage is that due to details of the dipole-dipoleinteraction, magnetic interactions directly above the NV centers areminimized, so the most sensitive interaction area is located somelateral distance away from each NV center. This isn't true for electricinteractions. The orientation also isn't ideal for optical excitationand readout using light totally internally reflected inside the diamond.However, it does represent a decent compromise between both and allowsuse of a diamond surface which is easier to manufacture.

FIG. 8B depicts another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings. To the configuration depicted in (A) is added a microwavesource 806, from which microwave radiation 802 is directed to thediamond surface by means of a microwave antenna 804. The microwavesource matches the zero field splitting frequency (2.89 GHz), and can beadded as in FIG. 8B in a direction that assures the angle of themicrowave magnetic field is the same relative to all the NV centers aswell to help the measurement accuracy. The intensity of fluorescentemission light 418 may be measured, and a resonance behavior of a colorcenter within the diamond may be identified based on a relationshipbetween the measured intensity and the frequency of microwave radiation802.

FIG. 8C depicts yet another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings. To the configuration depicted in (B) is added a magneticfield source 810, by which an external magnetic field 812 is imposed onthe diamond surface. Adding an external static field H₀ as indicated onFIG. 8C may be required for phase coherence (T₂) measurements.

FIG. 8D depicts still another illustrative configuration of a device fordetecting target molecules, according to aspects of the presentteachings. To the configuration depicted in (C) is added a secondmicrowave source 808 which shares the same microwave antenna 804 as thefirst microwave source 806. A second (independent frequency) microwavesource as shown in FIG. 8D may be required for the spin-labels toperform DEER experiments—note that all microwave magnetic fields areideally close to perpendicular to H₀. Because at high magnetic fields,only NV centers parallel with the field can be read out optically, thisconfiguration would ideally use a (111) surface with NV centers alignedalong the [111] direction as described above. If all NV centerorientations were populated equally, 75% of them would be unusable.Optical excitation and fluorescence emission are ideally close toparallel with the NV axes.

In the case of non-equivalent NV centers (like the (100) terminatedcrystals with all 8 different orientations populated) a possiblesolution is to use only ⅛ of the NV centers with the field properlyaligned relative to them, or to address them all sequentially byapplying different field orientations and microwave frequencies. Anotherpossible solution is to use what is known as “field cycling” in themagnetic resonance field. In this case, a large field (e.g., bigger thantwo times the zero field splitting) could be applied along the [100]axis during the microwave operations and switched to zero during opticalpolarization and detection operations.

FIG. 9 depicts still another illustrative configuration of a device 899for detecting target molecules, according to aspects of the presentteachings. More specifically, FIG. 9 is a schematic representation anintegrated biochip based on the invention (not to scale). Although thisfigure depicts an embodiment of the detection technique that utilizesspin labels, aptamers, and nitrogen vacancy centers, other embodimentsof the detection technique are possible. The biochip is comprised offour layers: the Capture Layer 900, the Diamond layer 902, the FilterLayer 904, and the Integrated Detection and Processing Layer 906. TheCapture Layer 900 is comprised of a channel that directs the samplefluid containing the target molecule 206 of interest (e.g., targetprotein 302) past the surface of the Diamond Layer, on which areattached SOMAmers 301, linked to magnetic spin labels 300.

Features in the Capture Layer are regions of the sensing surface of theDiamond Layer on which identical SOMAmers are attached. Each featurecontains one or more SOMAmers specific to a separate protein analyte,and corresponds in x-y coordinates (FIG. 9 ) to an associated collectionof NV centers in the Diamond Layer and to an individual photodetector inthe Integrated Detection and Processing Layer.

Each SOMAmer 301 or other capture reagent 200 may be a specific type orspecies of capture reagent configured to bind to a specific type orspecies of target molecule 206. The species of the capture reagent maybe associated with a functional group of the capture reagent. Capturelayer 900 may include capture reagents 200 belonging to at least twodifferent species, such that at least two species of target molecule 206may be detected by the biochip device. In some embodiments, Capturelayer 900 includes capture reagents from a large number of species,e.g., hundreds of species, thousands of species, or more.

Some preferred embodiments of the present invention do not include spinlabels. Binding of a protein molecule to a SOMAmer molecule results in achange in the interaction 304 between the SOMAmer and proximalnitrogen-vacancy centers 104, such changes being detected viafluorescent emission 216. The surface chemistry and SOMAmer areoptimized to create a large relative difference in interaction uponbinding of a protein target to the aptamer.

The Diamond Layer is comprised of high purity diamond fabricated bychemical vapor deposition, in which a layer of NV centers 104 isembedded at a prescribed fixed distance 105 from the surface. In someexamples, the prescribed fixed distance, which may also be referred toas a depth, is in a range of 5-20 nanometers, or 15-20 nanometers. TheNV centers within the Diamond Layer are separated by a distance muchgreater than the prescribed fixed depth, assuring the interactionsbetween them are small compared to interactions with molecules on thesurface. The thickness of the layer of NV centers is small compared tothe prescribed fixed depth. The distance between the nitrogen-vacancycenters and the SOMAmers is chosen to be sufficiently small that thechange in interaction upon binding the protein to the SOMAmer ispossible and/or detectable. An external magnetic field 812 may bepresent. The Diamond Layer may include 100% ¹²C diamond.

Excitation light 208 (produced by an optical source such as a laser)stimulates fluorescence of the NV centers. External microwaves 802transition ground-state electrons between their sublevels. The FilterLayer is comprised of layered dielectric materials to form a dichroicbandpass filter 912, which is adhered to the Diamond Layer 902 on oneside, and the Integrated Detection and Processing Layer 906 on the otherside. In addition to passing emitted light from the NV centers whileblocking all other wavelengths, the Filter Layer 904 also blocks emittedlight that is outside a narrow range of angles from normal, therebyminimizing crosstalk in detection between neighboring NV centers.

The Filter Layer is designed to be a narrow band-pass filter around theemission wavelength of the nitrogen-vacancy centers (689 nm). Inaddition, the Filter Layer blocks light outside of a narrow band ofangles from the normal, in order to minimize cross talk betweenneighboring features. The Filter Layer also serves as a mechanicallinkage between the Diamond Layer and the Integrated Detection andProcessing Layer. Optically clear and mechanically rigid adhesive may beused to join the Filter Layer to the Diamond Layer at the top, and tojoin the Filter Layer to the Integrated Detection and Processing Layerat the bottom. The Filter Layer is constructed of layers of dielectricmaterials that rely on wave interference principles to block certainwavelengths of light. The design and fabrication of the Filter Layerfalls outside the purview of the present teachings.

The Integrated Detection and Processing Layer 906 is comprised of CMOSavalanche photodetectors 914 which drive high-speed electronic gates 916which drive electronic event counters 918. Photodetectors 914 may alsobe referred to as a detector assembly. According to the presentteachings, a detector assembly may be configured to irradiate colorcenters with excitation light of one or more frequencies, and to detectemission of electromagnetic radiation from the color centers. The outputof the event counters is collected for analysis. The designatedcoordinates 920 are chosen such that the x and y coordinates lie in theplane of the upper Diamond Layer surface, and the z coordinate isperpendicular to the surface. The range of wavelengths emitted by theoptical source (i.e., the spectrum of excitation light 208) may or maynot be the same as the range of wavelengths detected by photodetectors914. The range of wavelengths of the excitation light may be partiallyoverlapping or substantially non-overlapping with the range ofwavelengths photodetectors 914 are configured to detect.

The Integrated Detection and Processing Layer may be designed forlithographic production, including both the electronic components andconnections between them. The Integrated Detection and Processing Layermay be fabricated using lithography and attached to the Filter Layer (orto the Diamond Layer, if the Filter Layer is omitted) using opticallytransparent adhesive. The Integrated Detection and Processing Layer maybe comprised of a photodetector layer, a gating layer, an A to D (analogto digital) layer, and a bus to move accumulated data to a processor.Although FIG. 9 depicts these layers as being distributed in thez-direction, such an arrangement is not necessary.

The photodetector layer may include an array of avalanche photodiodes,each photodiode dedicated to a single feature on the Capture Layer, andbeing associated with this feature in the x and y coordinates. Eachphotodiode may be associated with a high-speed electronic gate in thegating layer. Changes in fluorescence from the NV centers can be on thesub-microsecond scale, and so to monitor these changes in intensity itis necessary to collect light only for very short and defined timeframes. Each gate in turn is associated with an event counter in theevent-counter layer, which actually performs the photon counting. Thedata from each event counter is bussed to a processor for data analysis.

There are at least three examples of arrangements in which the featuresof the Capture Layer of FIG. 9 (i.e., regions of the sensing surface ofthe Diamond Layer on which SOMAmers are attached) could be constructed,each offering different advantages. In a first example, a single SOMAmeris associated with a single NV center, which is in turn associated witha single photodetector. Such a setup offers advantages in terms ofdetection, but requires precise control of the placement of the NVcenter or of single SOMAmers. The primary advantage of this mode ofconstruction is that it offers detection of molecules on an individualbasis, which allows a third dimension of specificity (after bindingaffinity and washing) based on binding characteristics which would belost in ensemble detection. Additionally, target molecules can becounted one-by-one eliminating some calibration steps. The primarydisadvantage of this mode is that many such features are required inorder to discriminate between different concentrations. Resolving ordersof magnitude of differences in concentration may require hundreds orthousands of such features for a single analyte (depending on thedesired level of resolution), increasing the complexity of the device.

The second illustrative mode of construction comprises a collection ofmultiple SOMAmers of the same type associated with a collection ofmultiple NV centers, which is in turn associated with a singlephotodetector. The random distribution of NV centers in the x-y plane(as defined in FIG. 9 ), and the random distribution of SOMAmers in thefeature collection, lead to an averaged signal at the photodetector,which should be more stable. A range of concentrations could also bequantified with a single photodetector, due to the multiple SOMAmers.However, using such collections instead of single SOMAmers or NV centersprohibits the third dimension of specificity allowed by the first modeand requires generation of calibration curves to correlate NV centerresponse to the concentration of the target protein in the fluid.

The third mode of construction is a hybrid of the first and second mode,comprising a collection of multiple SOMAmers of the same type beingassociated with a single NV center, which is in turn associated with asingle photodetector. As with the second mode, a range of concentrationscould be quantified with a single photodetector, due to the multipleSOMAmers, which also lead to an averaged signal. The use of a single NVcenter may lead to a cleaner signal. Randomly distributed NV centers maybe optically located in the x-y plane, followed by placement of SOMAmercollections at the same location.

FIG. 10 depicts steps performed in an illustrative method 1000 fordetecting target molecules in a sample fluid, and may not recite thecomplete process or all steps of the method. Although various steps ofmethod 1000 are described below and depicted in FIG. 10 , the steps neednot necessarily all be performed, and in some cases may be performedsimultaneously, or in a different order than the order shown.

At step 1002, the method includes contacting a capture reagent with asample fluid. The capture reagent is attached to a surface (e.g., asurface of a crystalline film or substrate, or a single-crystal diamond)and is configured to bind to a desired target molecule. The capturereagent may be described as captured by the surface, bound to thesurface, attached to the surface, tethered to the surface and/orimmobilized on the surface. The target molecule may be any targetmolecule having the ability to bind with a capture reagent, includingbut not limited to target molecules described in this disclosure, suchas a protein. Similarly, the capture reagents may be any suitablecapture agents. This includes but is not limited to any of the capturereagents described in this disclosure, such as aptamers, nucleic acidmolecules, or nucleic acid molecules having at least one 5-positionmodified pyrimidine (such as SOMAmers). In some examples, at least twodifferent 5-position modified pyrimidines are attached to the surface,including at least one 5-position modified uridine and at least one5-position modified cytidine.

At step 1004, the method includes irradiating a color center disposedproximate the surface (e.g., at a fixed depth within the crystallinefilm) with excitation light. The excitation light is configured (e.g.,through selection of central wavelength or bandwidth) to inducefluorescent emission by the color center. In some examples, the colorcenter is a nitrogen-vacancy center of a diamond crystal, and thesurface is a surface of the diamond crystal.

At step 1006, the method includes measuring the intensity of thefluorescent emission using one or more detectors (e.g., thephotodetectors of the Integrated Detection and Processing Layerdescribed above with reference to FIG. 9 ).

At step 1008, the method includes detecting a change in the intensity ofthe fluorescent emission. The intensity changes in response to binding atarget molecule to a capture reagent, and therefore changes in theemitted intensity indicate the presence of a target molecule.

Optionally, at step 1010, the method may include irradiating the colorcenter with microwave radiation at a frequency capable of inducingconversion of ground-state electrons in the color center from a firstsub-state to a second sub-state. For example, the microwave radiationmay include a resonant frequency corresponding to an energy differencebetween a zero-spin sub-state of the ground state and a non-zero-spinsub-state of the ground state. Detecting a change in the intensity ofthe fluorescent emission in response to binding of the target moleculeat step 1008 may further include identifying resonance behavior of thecolor center based on a relationship between the measured intensity ofthe fluorescent emission and the frequency of the microwave radiation.For example, the binding of the target molecule may induce a change inthe resonant frequency between the first and second sub-states. Thechange in the resonant frequency may be identified based on thefluorescent emission intensity measured while varying the frequency ofthe microwave radiation.

FIG. 11 depicts steps performed in an illustrative method 1100 formeasuring a concentration of target molecules, and may not recite thecomplete process or all steps of the method. Although various steps ofmethod 1100 are described below and depicted in FIG. 11 , the steps neednot necessarily all be performed, and in some cases may be performedsimultaneously, or in a different order than the order shown.

At step 1102, the method includes exposing a fluid to the surface of acrystalline film (e.g., a diamond film, among others) to allow capturereagents attached to the surface to bind to target molecules (e.g.,proteins, among others) within the fluid. The crystalline film includesat least one color center, such as a nitrogen-vacancy center or othersuitable defect.

At step 1104, the method includes irradiating the crystalline film withexcitation light configured to induce fluorescent emission from at leastone color center within the film. Optionally, step 1104 includesirradiating the color center with microwave radiation having a frequencycapable of inducing conversion of ground-state electrons in the colorcenter from a first sub-state to a second sub-state.

At step 1106, the method includes detecting a change in a property ofthe color center based on the fluorescent emission. The change in thecolor center property arises in response to binding of the targetmolecule by the capture reagent. In some examples, the capture reagentsinclude a magnetic spin label, such that binding between the targetmolecule and the capture reagent changes a magnetic field at the colorcenter. The change in the color center property may be at leastpartially caused by the change in the magnetic field.

At step 1108, the method includes determining a concentration of targetmolecules within the fluid based on the detected change in the colorcenter property. For example, characteristics of the detected change mayindicate the number or approximate number of target molecules bound bythe capture reagents to the surface of the film, and the concentrationof target molecules within the fluid can be deduced from the number oftarget molecules and the volume of the fluid.

FIG. 12 depicts steps performed in an illustrative method 1200 fordetecting target molecules, and may not recite the complete process orall steps of the method. Although various steps of method 1200 aredescribed below and depicted in FIG. 12 , the steps need not necessarilyall be performed, and in some cases may be performed simultaneously, orin a different order than the order shown.

At step 1202, the method includes exposing a crystalline substrate(e.g., a diamond film) to a sample fluid, such that capture reagentsattached to the surface of the substrate can bind to target moleculesexpected to be present in the sample fluid. The crystalline substrateincludes at least one color center, such as a nitrogen-vacancy center indiamond. The target molecules may be proteins or any other targetmolecule described elsewhere in this disclosure. Likewise, the capturereagents may be any molecules suitable for binding specifically to thedesired target molecules, and may include aptamers, SOMAmers, or thelike. In some examples, the capture reagents are aptamers including atleast two distinct 5-position modified pyrimidines.

At step 1204, the method includes irradiating the crystalline substratewith electromagnetic radiation. The electromagnetic radiation isconfigured to stimulate or induce fluorescence by color centers withinthe crystalline substrate. Characteristics of the electromagneticradiation, including central frequency, bandwidth, intensity, pulseenergy, pulse duration, and/or polarization may be designed to at leastpartially determine characteristics of the emitted fluorescence.

At step 1206, the method includes detecting fluorescence emitted by thecolor center, e.g., by one or more photodetectors.

At step 1208, the method includes identifying a change in a property ofthe one or more color centers caused by binding one or more targetmolecules from the sample fluid to the capture reagents. For example,identifying the change may include identifying a change in the spectrumof radiation emitted by the color centers, such as detecting fluorescentradiation emitted by the color centers, and may include identifyingchanges in an intensity, temporal variation, or spectrum of the emittedfluorescence.

FIG. 13 depicts steps in an illustrative method 1300 for manufacturing adevice for detecting the presence of protein molecules in a samplefluid, and may not recite the complete process or all steps of themethod. Although various steps of method 1300 are described below anddepicted in FIG. 13 , the steps need not necessarily all be performed,and in some cases may be performed simultaneously or in a differentorder than the order shown. Aspects of method 1300 may, for example, beused to manufacture the integrated biochip depicted in FIG. 9 .

At step 1302, the method includes fabricating a crystalline film, e.g.,by chemical vapor deposition. For example, ¹²C-enriched diamond filmsmay be produced by plasma-enhanced chemical vapor deposition of amixture of methane and hydrogen gas on diamond substrates.

At step 1304, the method includes embedding substitute atoms in thecrystalline film. The substitute atoms are suitable for collocating withvacancies in the crystalline film to form color centers.

At step 1306, the vacancies are created in the crystalline film, e.g.,by impinging an ion beam and/or electron beam on the film.

At step 1308, the method includes creating color centers in thecrystalline film by collocating at least some of the substitute atomswith at least some of the vacancies. Collocating the substitute atomsand the vacancies may include, for example, annealing the film at a hightemperature, or by any other suitable process.

At step 1310, the method includes attaching capture reagents to a firstsurface of the crystalline film. Step 1310 may include attaching capturereagents to some regions of the surface of the crystalline film, andpassivating regions of the surface not bound with capture reagentsagainst non-specific binding by undesired proteins. Attaching thecapture reagents and passivating other regions of the surface may beaccomplished by binding passivating molecules (e.g., hydrophilicpolymers) to the surface, with some of the passivating moleculesincluding active groups for linkage to specific target molecules. Forexample, some of the passivating molecules may have a first endconfigured to bind to the surface, and a second end having an activegroup configured to bind to DNA or other target molecules.

At step 1312, the method includes attaching photodetectors to a secondsurface of the crystalline film, such that the photodetectors arecapable of detecting emission from the color centers. The photodetectorsmay be fabricated using lithography, and may be adhered to thecrystalline film using optically transparent, mechanically rigidadhesive. In some examples, a filter layer (e.g., Filter Layer 904) isincluded between the photodetectors and the film.

Typical detection schemes for measuring target molecule concentration ina sample fluid include measuring an intensity of fluorescent emission byat least one color center, identifying a change in the fluorescentemission in response to binding of at least one target molecule to acapture reagent, and determining a concentration of target moleculeswithin a sample fluid based on the identified change. For example,aspects of the nitrogen-vacancy center excitation and emission depictedin FIG. 7 may be included in an illustrative method where the relaxationtime, T₁, is monitored by purely optical methods. T₁ is measured bypolarizing the ground spin states to the m_(s)=0 sublevel by irradiatingthe NV centers with excitation light. As shown in FIG. 4 , a fraction ofthe electrons excited from the m_(s)=±1 sublevels return to the groundstate via the dark transition, thereby undergoing a transition to them_(s)=0 sublevel, which provides maximum intensity. T₁ is determined byperiodically monitoring the fluorescent intensity after polarization viaexcitation has been discontinued and seeing how fast the ground statereturns to a fully unpolarized state. An illustrative method formeasuring protein in urine includes the following steps, which may beperformed simultaneously or in a different order than the order shown:

-   -   1. Prepare multiple calibration samples of urine containing        known concentrations of the protein of interest,    -   2. Flow these calibration samples across the sensing surface of        the diamond layer, allowing the protein sufficient opportunity        to bind to the attached SOMAmers, optionally recirculating the        sample fluid,    -   3. Flow wash fluid across the fluid-contacting surface of the        diamond layer, allowing the wash fluid sufficient opportunity to        remove molecules binding in non-specific fashion to the        SOMAmers, optionally recirculating the wash fluid,    -   4. Irradiate the NV centers with a series of pulses of        excitation light, each pulse of long enough duration to cause        full polarization of the NV center into the m_(s)=0 state. The        time spacing T₁ between these excitation pulses is varied,    -   5. Measure the fluorescent intensity immediately after the start        of each excitation pulse, as well as a reference intensity,        taken towards the end of each excitation pulse when the spin        states should be fully polarized to the m_(s)=0 sublevel,    -   6. Calculate normalized intensities by dividing the fluorescent        intensity at the start of each excitation pulse with the        corresponding reference intensity,    -   7. Generate response plots for each of the calibration samples,        by plotting the reference intensities for each pulse p_(i)        against the spacing time T₁ immediately preceding pulse p_(i),    -   8. Find the relaxation time T₁ for each calibration sample by        fitting a decaying exponential of the form normalized        intensity=I₀*exp(−τ/T₁).    -   9. Generate a calibration curve of protein concentration versus        T₁,    -   10. Repeat steps (2) through (8), using samples of interest        instead of calibration samples to generate response plots, and    -   11. Apply the calibration curve of step 9 to determine the        protein concentration of each sample of interest.

Additional methods may involve, for example, systems shown in FIG. 8 .While optical detection of the spin state is preferred, the addition ofmicrowave excitation and the introduction of a static field open up allthe techniques from traditional EPR for manipulating spin states.Examples of those (e.g., Hahn echoes, DEER, etc.) have been describedabove. There are a huge number of variations on the method above whichadd additional spin manipulation steps via techniques well known in theart that can be chosen to optimize detection of the bound analytes.

While the examples given have focused mainly on urine or blood (serum orplasma) as sample fluids, biochips according to the present teachingscould work on a variety of biological fluids or solutions derived fromthem, for example, by dilution. These include blood, plasma, serum,urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid,lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, jointaspirate, leukocytes, peripheral blood mononuclear cells, sputum,breath, cells, a cellular extract, stool, tissue, a tissue extract, atissue biopsy, and cerebrospinal fluid.

Illustrative Combinations and Additional Examples

This section describes additional aspects and features of proteomicassays, presented without limitation as a series of paragraphs, some ofall of which may be alphanumerically designated for clarity andefficiency. Each of these paragraphs can be combined with one or moreother paragraphs, and/or with disclosure from elsewhere in thisapplication, including any material incorporated by reference, in anysuitable manner. Some of the paragraphs below expressly refer to andfurther limit other paragraphs, providing without limitation examples ofsome of the suitable combinations.

A. A device for detection of one or more species of target molecules ina fluid comprising:

-   -   a) A solid support with a surface; and    -   b) One or more species of capture reagents attached to the        surface;    -   c) Wherein each species of capture reagent binds selectively to        a particular species of target molecule; and    -   d) Color centers located close to the surface of the solid        support; and    -   e) A detector or detectors for detecting changes in the        properties of the color centers upon binding of protein        molecules to the capture reagents.

A1. The device of paragraph A, wherein the fluid contains biologicalfluids and the target molecules are proteins.

A2. The device of paragraph A1, wherein the capture reagents areaptamers.

A3. The device of paragraph A2, wherein the aptamers are nucleic acidmolecules.

A4. The device of paragraph A3, wherein at least some of the nucleicacid molecules have at least one 5-position modified pyrimidine.

A5. The device of paragraph A4, wherein the solid support is a diamondcrystal and the color centers are nitrogen-vacancy centers.

A6. The device of paragraph A5, wherein the detector or detectors is anoptical system that irradiates the nitrogen-vacancy centers with a firstrange of wavelengths of radiation and detects a second range ofwavelengths.

A7. The device of paragraph A6, wherein the optical source is a pulsedoptical source.

A8. The device of paragraph A7, wherein the changes in properties of thenitrogen vacancy centers are changes in the magnetic resonanceproperties of the nitrogen vacancy centers.

A9. The device of paragraph A8, further comprising a magnetic fieldsource to provide a magnetic field.

A10. The device of paragraph A9, further comprising a microwave sourceto provide microwave radiation.

A11. The device of paragraph A10, wherein the microwave source is tunedto a frequency resonant with the sublevels of the nitrogen vacancycenter electronic ground state.

A12. The device of paragraph A11, wherein the microwave source is apulsed source.

A13. The device of paragraph A5, wherein the surface of the solidsupport is a {111} surface of a single crystal diamond.

A14. The device of paragraph A5, wherein the surface of the solidsupport is a {100} surface of a single crystal diamond.

A15. The device of paragraph A5, wherein the nitrogen vacancy centersare located within 25 nanometers of surface of the diamond crystal.

A16. The device of paragraph A7, wherein the nucleic acid moleculesadditionally include a spin label.

A17. The device of paragraph A12, wherein the nucleic acid moleculesadditionally include a spin label.

B. A device for simultaneously quantifying single or multiple analytesin a sample fluid comprising:

-   -   a) A thin crystalline layer, one surface which makes contact        with the sample fluid, containing color center defects 5-25        nanometers from the fluid-contacting surface;    -   b) Capture agents that are attached to the fluid-contacting        surface of the crystalline layer in close proximity to the color        center defects described in (a), such that binding of analytes        to the binding agents affect the local magnetic field external        to the color center defects thus detectably changing the        behavior of said color center defects; and    -   c) A means of detecting the said change in behavior of said        color center defects.

B1. The device of paragraph B, in which the said change in behavior ofsaid color center defects includes a change in fluorescence and the saidmeans of detecting this change in fluorescence comprises:

-   -   a) An optical filter layer bonded to the non-contacting surface        of the crystalline layer, for passing fluorescent emissions from        the color center defects while excluding excitation light or        other light;    -   b) A detection layer, bonded to the optical filter layer on the        side opposite of the crystalline layer, for the capture and        quantitation of the fluorescent light emitted by the color        center defects, including the intensity; and    -   c) A means for introducing excitation light into the crystalline        layer, such that the color center defects are stimulated to emit        fluorescent light;

B2. The device of paragraph B1, in which the said means for introducingexcitation light into the crystalline layer does such introduction oflight through the edges of the said crystalline layer, such that thesaid excitation light travels in a direction parallel to the saidfluid-contacting surface of the crystalline layer.

B3. The device of paragraph B, in which the said change in behavior ofsaid color center defects includes a change in fluorescence and the saidmeans of detecting this change in fluorescence comprises:

-   -   a) An optical wave guide, optionally including an optical        filter, bonded to the non-contacting surface of the crystalline        layer, for passing fluorescent emissions from the color center        defects while excluding excitation light or other light;    -   b) A detection layer, situated at the opposing end of the        optical wave guide from the said crystalline layer, for the        capture and quantitation of the fluorescent light emitted by the        color center defects, including the intensity; and    -   c) A means for introducing excitation light into the crystalline        layer, such that the color center defects are stimulated to emit        fluorescent light.

B4. The device of paragraph B, in which the said change in behavior ofsaid color center defects includes a change in the electric field ormagnetic field local to the said color center defect and the said meansof detecting this said change in electric field or magnetic fieldcomprises an electronic or opto-electronic detector.

B5. The device of paragraph B1, further comprising a means forintroducing microwave radiation into the crystalline layer, in order toaffect and/or monitor the resonance behavior of the color centerdefects.

B6. The device of paragraph B5, in which is included a means forimposing a constant or variable magnetic field across the regionscontaining color center defects in order to modify their resonancebehavior.

B7. The device of paragraph B5, wherein the said thin crystalline layeris a diamond film, and the said color center defects arenitrogen-vacancy centers.

B8. The device of paragraph B7, wherein the said device may beregenerated for multiple uses.

B9. The device of paragraph B8, wherein the said analyte-specificbinding agents are linked to magnetic spin labels to increase the effectupon binding of said analytes on the local magnetic field external tothe color center defects thus detectably changing the behavior of saidcolor center defects.

B10. The device of paragraph B9, wherein the said analyte-specificbinding agents are aptamers or SOMAmers.

B11. The device of paragraph B9, wherein the said optical filter layeris comprised of layered dielectric films to form a dichroic bandpassfilter.

B12. The device of paragraph B9, wherein the said detection layer iscomprised of CMOS avalanche photodetectors in immediate contact with thesaid optical filter layer, wherein the signals from said photodetectorsare passed through high-speed electronic gates, the signals that passthrough said high-speed gates are passed to event counters, and theresulting data collected by the event counters are routed to a processorfor analysis.

B13. The device of paragraph B9, in which a collection of said tetheredSOMAmers are identical in their specificity for a specific analyte andinteract in a measurable way with a collection of said nitrogen-vacancycenters which collectively emit fluorescent light captured by anindividual photodetector.

B14. The device of paragraph B13, in which the said collection ofSOMAmers consists of a single molecule, and the said collection ofnitrogen-vacancy centers consists of a single center.

B15. The device of paragraph B13, in which the said collection ofSOMAmers consists of a multiple molecules identically specific for thesame analyte, and the said collection of nitrogen-vacancy centersconsists of a single center.

B16. The device of paragraph B13, in which there are multiple saidcollections of tethered SOMAmers, each said collection being specific toa different analyte.

B17. The device of paragraph B16, in which the number of saidcollections of tethered SOMAmers is between 100 and 10,000.

B18. The device of paragraph B13, in which means are included to directfluids to the fluid-contacting surface of the diamond layer, includingmeans to recirculate said fluids if desired, said fluids to include:

-   -   a) The sample fluid;    -   b) Wash fluid used to remove molecules binding in non-specific        fashion to the said SOMAmers or the said fluid-contacting        surface of the diamond layer; and    -   c) Regeneration fluid used to remove molecules binding in both        specific and non-specific fashion to the said SOMAmers or the        said fluid-contacting surface of the diamond layer without        causing damage or denaturation to said SOMAmers or said        fluid-contacting surface of the diamond layer.

B19. The device of paragraph B18, in which the said sample fluid is abiological fluid from a human subject.

B20. The device of paragraph B19, in which the said biological fluid isurine.

B21. The device of paragraph B20, in which the device is designed to becontained within a toilet, urinal, or other urine receptacle, therebyallowing the capture and analysis of urine from said toilet, urinal, orurine receptacle.

B22. The device of paragraph B21, in which the data generated by thedevice is compared with a proteomic database for diagnosis of possibledisease states.

B23. The device of paragraph B18, in which analyte concentrations aremeasured in the said sample fluid according to the described method:

-   -   a) Preparing multiple calibration samples of fluids containing        known concentrations of the said analyte;    -   b) Directing said calibration samples to the said        fluid-contacting surface of the diamond layer and allowing the        said analyte sufficient opportunity to bind to the said tethered        SOMAmers, optionally including recirculation of said calibration        sample fluid;    -   c) Directing said wash fluid to the said fluid-contacting        surface of the diamond layer, allowing the said wash fluid        sufficient opportunity to remove molecules binding in        non-specific fashion to the said SOMAmers, such opportunity        optionally including recirculation of said wash fluid;    -   d) Irradiating the said nitrogen-vacancy centers with excitation        light, said light including frequencies that induce fluorescent        emission of said nitrogen-vacancy centers, and measuring said        fluorescent emission intensity;    -   e) Irradiating the said nitrogen-vacancy centers with microwave        radiation, the frequency of said microwave radiation being        varied across a range that is expected to include resonant        frequencies that induce conversion of ground state electrons in        the said nitrogen-vacancy centers from the 0-spin state to the        ±1-spin states;    -   f) Generating plots for each said calibration sample of said        fluorescent emission intensity versus said microwave radiation        frequency;    -   g) Generating a calibration curve of analyte concentration        versus some chosen characteristic of the plots of said        calibration samples from (f);    -   h) Repeating steps (b) through (f), using samples of interest        instead of calibration samples to generate plots as in (f); and    -   i) Applying the said calibration curve of step (f) to determine        the analyte concentration of each said sample of interest.

B24. The device and method of paragraph B23, in which the said chosencharacteristic of the said plot is the resonant microwave frequency,determined by locating the most extreme local minimum.

B25. The device and method of paragraph B23, in which the said chosencharacteristic of the said plot describes the width of the resonantinverted peak in some fashion, for instance the full-width-half-maximum(FWHM), or the coefficient of variation (CV).

B26. The device of paragraph B18, in which analyte concentrations aremeasured in the said sample fluid according to the described method:

-   -   a) Preparing multiple calibration samples of fluids containing        known concentrations of the said analyte;    -   b) Directing said calibration samples to the said        fluid-contacting surface of the diamond layer and allowing the        said analyte sufficient opportunity to bind to the said tethered        SOMAmers, optionally including recirculation of said calibration        sample fluid;    -   c) Directing said wash fluid to the said fluid-contacting        surface of the diamond layer, allowing the said wash fluid        sufficient opportunity to remove molecules binding in        non-specific fashion to the said SOMAmers, such opportunity        optionally including recirculation of said wash fluid;    -   d) Irradiating the said nitrogen-vacancy centers with a series        of square wave pulses, p_(i), of excitation light, said light        including frequencies that induce fluorescent emission of said        nitrogen-vacancy centers, each said pulse of long enough        duration to cause full polarization of the said nitrogen-vacancy        centers into the 0-spin state. Further varying the spacing τ_(i)        between these said excitation pulses p_(i);    -   e) Measuring the fluorescent intensity immediately after the        start of each said excitation pulse, as well as a reference        intensity, taken towards the end of each said excitation pulse,        said reference intensity to be measured when the spin states        should be fully polarized to the 0-spin state;    -   f) Calculating normalized intensities by dividing the said        fluorescent intensity at the start of each said excitation pulse        p_(i) with the corresponding said reference intensity;    -   g) Generating response plots for each of the said calibration        samples, by plotting the said reference intensities for each        said pulse p_(i) against the said spacing time τ_(i) immediately        preceding pulse p_(i);    -   h) Finding the relaxation time T₁ for each said calibration        sample by fitting a decaying exponential of the form        y=I₀*exp(−τ/T₁), where y is the dependent variable consisting of        the collection of said normalized intensities for each said        pulse p_(i), and the independent variable T is the collection of        said spacing times preceding each said pulse p_(i);    -   i) Generating a calibration curve of known analyte concentration        in the said calibration samples versus relaxation time T₁ from        step (h);    -   j) Repeating steps (b) through (h), using samples of interest        instead of calibration samples to generate plots as in (g) and        finding relaxation times as in step (h); and    -   k) Applying the said calibration curve of step (i) to determine        the analyte concentration of each said sample of interest.

C. A device for detecting target molecules, comprising:

a surface configured to contact a fluid;

a plurality of capture reagents attached to the surface, each capturereagent configured to bind to a target molecule;

a plurality of color centers located proximate the surface; and

at least one detector configured to detect a change in a property of atleast one of the color centers in response to binding the targetmolecule to one of the capture reagents.

C1. The device of paragraph C, wherein the target molecule is a protein,and the capture reagents are aptamers.

C2. The device of paragraph C1, wherein the aptamers are nucleic acidmolecules.

C3. The device of paragraph C2, wherein the nucleic acid molecules haveat least one 5-position modified pyrimidine.

C4. The device of paragraph C, wherein the surface is a surface of adiamond crystal, and the color centers are nitrogen-vacancy centers ofthe diamond crystal.

C5. The device of paragraph C4, wherein the diamond crystal is asingle-crystal diamond.

C6. The device of paragraph C4, further comprising an optical sourceconfigured to irradiate the nitrogen-vacancy centers with radiationhaving a first range of wavelengths, and wherein the detector isconfigured to detect radiation having a second range of wavelengths.

C7. The device of paragraph C4, wherein the property is associated witha magnetic resonance of the nitrogen vacancy centers.

C8. The device of paragraph C4, further comprising a microwave sourceconfigured to provide microwave radiation having a range of frequenciesincluding a resonant frequency of sublevels of an electronic groundstate of the nitrogen vacancy centers.

C9. The device of paragraph C, wherein capture reagents includesreagents belonging to a plurality of capture species, and each capturespecies is configured to bind to target molecules of a particular targetspecies.

C10. A device for measuring a concentration of target molecules,comprising:

a crystalline film including at least one color center;

a plurality of capture reagents attached to a surface of the crystallinefilm and configured to bind to a target molecule; and

a detector assembly configured to irradiate the color center withexcitation light and to detect emission of electromagnetic radiationfrom the color center.

C11. The device of paragraph C10, wherein the capture reagents include amagnetic spin label, and a magnetic field at the color center changes inresponse to binding the target molecule to one of the capture reagents.

C12. The device of paragraph C11, wherein the detector assembly isconfigured to irradiate the color center with microwave radiation havinga frequency capable of inducing conversion of ground-state electrons inthe color center from a first sub-state to a second sub-state.

C13. The device of paragraph C10, wherein the crystalline film is adiamond film, and the color center is a nitrogen vacancy center.

C14. The device of paragraph C10, wherein binding of one of the capturereagents to one of the target molecules produces a detectable change inthe emission of electromagnetic radiation from the color center bychanging an interaction between a spin label of the capture reagent andthe color center.

C15. A device for detecting target molecules, comprising:

a plurality of capture reagents attached to a surface of a crystallinesubstrate and configured to capture target molecules from a samplefluid;

a plurality of color centers disposed at fixed distances from thecapture reagents, the fixed distances being sufficiently small that aproperty of at least one of the color centers changes in response tocapture of one of the target molecules by one of the capture reagents;and

a detector configured to detect a change in the property of at least oneof the color centers.

C16. The device of paragraph C15, wherein the capture reagents areaptamers.

C17. The device of paragraph C16, wherein the capture reagents areoligonucleotides.

C18. The device of paragraph C16, wherein the capture reagents arenucleic acid molecules having at least one 5-position modifiedpyrimidine.

C19. The device of paragraph C18, wherein the crystalline substrate is adiamond film, and the color centers are nitrogen vacancy centers.

D0. A method for detecting target molecules in a sample fluid, themethod comprising:

contacting, with a sample fluid, a capture reagent attached to a surfaceand configured to bind to a target molecule;

irradiating a color center disposed proximate the surface withexcitation light configured to induce fluorescent emission by the colorcenter;

measuring an intensity of the fluorescent emission using one or moredetectors; and

detecting a change in the intensity of the fluorescent emission inresponse to binding the target molecule to the capture reagent.

D1. The method of D0, further comprising:

irradiating the color center with microwave radiation at a frequencycapable of inducing conversion of ground-state electrons in the colorcenter from a first sub-state to a second sub-state; and

wherein detecting a change in the intensity of the fluorescent emissionin response to binding the target molecule to the capture reagentincludes identifying resonance behavior of the color center based on arelationship between the measured intensity of the fluorescent emissionand the frequency of the microwave radiation.

D2. The method of paragraph D0 or D1, wherein the target molecule is aprotein.

D3. The method of any of paragraphs D0-D2, wherein the capture reagentsare aptamers.

D4. The method of any of paragraphs D0-D3, wherein the capture reagentsare nucleic acid molecules having at least one 5-position modifiedpyrimidine.

D5. The method of any of paragraphs D0-D4, wherein the surface is asurface of a diamond crystal, and the color centers are nitrogen-vacancycenters of the diamond crystal.

D6. The method of any of paragraphs D0-D5, wherein the capture reagentsare aptamers including at least one first 5-position modified pyrimidineand at least one second 5-position modified pyrimidine, wherein thefirst 5-position modified pyrimidine and the second 5-position modifiedpyrimidine are different 5-position modified pyrimidines;

wherein the first 5-position modified pyrimidine is a 5-positionmodified uridine and

wherein the second 5-position modified pyrimidine is a 5-positionmodified cytidine; or

wherein the first 5-position modified pyrimidine is a 5-positionmodified cytidine and

wherein the second 5-position modified pyrimidine is a 5-positionmodified uridine.

E0. A method for measuring a concentration of target molecules,comprising:

exposing a fluid to a surface of a crystalline film to allow capturereagents attached to the surface to bind to target molecules within thefluid;

irradiating the film with excitation light configured to inducefluorescent emission by at least one color center within the crystallinefilm;

detecting, based on the fluorescent emission, a change in a property ofthe color center in response to binding between the target molecules andthe capture reagents; and

determining, based on the detected change, a concentration of targetmolecules within the fluid.

E1. The method of paragraph E0, wherein the target molecules areproteins.

E2. The method of paragraph E1 or E2, wherein the capture reagentsinclude a magnetic spin label, and a magnetic field at the at least onecolor center changes in response to binding between the target moleculesand the capture reagents.

E3. The method of any of paragraphs E0-E2, wherein irradiating the filmwith excitation light includes irradiating the color center withmicrowave radiation having a frequency capable of inducing conversion ofground-state electrons in the color center from a first sub-state to asecond sub-state.

E4. The method of any of paragraphs E0-E3, wherein the color center is anitrogen vacancy center.

E5. The method of any of paragraphs E0-E4, wherein the crystalline filmis a diamond film.

F0. A method for detecting target molecules comprising:

exposing a crystalline substrate to a sample fluid such that a pluralityof capture reagents attached to a surface of the crystalline substratebind to target molecules within the sample fluid; and

identifying a change in a property of one or more color centers withinthe crystalline substrate in response to binding the target molecules tothe capture reagents.

F1. The method of paragraph F0, wherein the crystalline substrate is adiamond film, and the color centers are nitrogen vacancy centers withinthe diamond film.

F2. The method of paragraph F0 or F1, further comprising irradiating thecrystalline substrate with electromagnetic radiation, and whereinidentifying a change in a property of one or more color centers includesdetecting fluorescent radiation emitted by the color centers.

F3. The method of any of paragraphs F0-F2, wherein the capture reagentsare aptamers including at least two distinct 5-position modifiedpyrimidine.

F4. The method of any of paragraphs F0-F3, wherein the target moleculesare proteins.

G0. A method for manufacturing a device for detecting the presence ofprotein molecules in a sample fluid, comprising:

fabricating a crystalline film;

embedding a plurality of substitute atoms within the crystalline film;

creating a plurality of vacancies within the crystalline film;

creating color centers within the crystalline film by collocating atleast some of the substitute atoms with at least some of the vacancies;

attaching a plurality of capture reagents to a first surface of thecrystalline film; and

attaching a layer of photodetectors to a second surface of thecrystalline film.

G1. The method of paragraph G0, wherein the crystalline film isfabricated using chemical vapor deposition.

G2. The method of paragraph G0 or G1, wherein the vacancies are createdusing an electron beam.

G3. The method of any of paragraphs G0-G2, wherein the substitute atomsare collocated with the vacancies by annealing the crystalline film at ahigh temperature.

G4. The method of any of paragraphs G0-G3, wherein attaching the layerof photodetectors includes fabricating the layer of photodetectors usinglithography and adhering the layer of photodetectors to the secondsurface of the crystalline film using optically transparent adhesive.

What is claimed is:
 1. A method for measuring a concentration oflabel-free target molecules in a sample fluid, the method comprising:exposing a substrate to a sample fluid such that a plurality of capturereagents attached to a surface of the substrate contact the samplefluid, the plurality of capture reagents including first capturereagents configured to bind to first label-free target molecules andsecond capture reagents configured to bind to second label-free targetmolecules different from the first label-free target molecules;detecting, based on emission of electromagnetic radiation from at leastone color center within the substrate, a change in a property of the atleast one color center in response to binding between the firstlabel-free target molecules and the first capture reagents; anddetermining, based on the detected change, a concentration of the firstlabel-free target molecules within the sample fluid.
 2. The method ofclaim 1, wherein the first and second label-free target molecules areproteins and the first and second capture reagents are aptamers.
 3. Themethod of claim 2, wherein the aptamers each have at least one5-position modified pyrimidine.
 4. The method of claim 1, wherein thesubstrate comprises a diamond crystal and the at least one color centeris a nitrogen-vacancy center of the diamond crystal.
 5. The method ofclaim 4, wherein the property is associated with a magnetic resonance ofthe nitrogen-vacancy center.
 6. The method of claim 1, furthercomprising irradiating the at least one color center with radiationhaving a first range of wavelengths configured to induce the emission ofthe electromagnetic radiation, and wherein the emitted electromagneticradiation has a second range of wavelengths nonidentical to the firstrange.
 7. The method of claim 6, further comprising irradiating the atleast one color center with microwave radiation having a range offrequencies including a resonant frequency of sublevels of an electronicground state of the at least one color center.
 8. The method of claim 1,wherein the first capture reagents each include a magnetic spin label.9. The method of claim 1, wherein the surface includes an array ofdiscrete regions each containing no more than one of the first capturereagents, and the at least one color center includes a plurality ofcolor centers each associated with exactly one of the first capturereagents.
 10. A method for detecting label-free target analytes in asample fluid, the method comprising: exposing to a sample fluid at leasttwo capture reagents attached to a surface, wherein each capture reagentis configured to bind to a different label-free target analyte, and thesurface is disposed proximate a plurality of color centers; and sensinga change in a property of a first color center of the plurality of colorcenters in response to binding one of the different label-free targetanalytes to a corresponding one of the capture reagents.
 11. The methodof claim 10, wherein sensing the change includes detecting, by aphotodetector, emission of electromagnetic radiation from the firstcolor center.
 12. The method of claim 11, wherein the photodetector isconfigured to detect emission from only the first color center of theplurality of color centers.
 13. The method of claim 11, wherein thecapture reagents are aptamers.
 14. The method of claim 13, wherein theaptamers each include a magnetic spin label.
 15. A method for detectinglabel-free target molecules, the method comprising: capturing a firstlabel-free target molecule from a sample fluid by a first capturereagent of a plurality of capture reagents attached to a surface,wherein the first capture reagent belongs to a first species configuredto bind to the first label-free target molecule, and the plurality ofcapture reagents further includes a second capture reagent belonging toa second species configured to bind to a second label-free targetmolecule; and sensing, based on emission of first electromagneticradiation from a color center disposed adjacent the surface, the captureof the first label-free target molecule by the first capture reagent.16. The method of claim 15, wherein the first and second label-freetarget molecules are proteins and the first and second capture reagentsare aptamers.
 17. The method of claim 16, wherein the first capturereagent has a functionalized 5-position pyrimidine base.
 18. The methodof claim 15, further comprising causing the emission of the firstelectromagnetic radiation from the color center by exciting the colorcenter by second electromagnetic radiation.
 19. The method of claim 15,wherein sensing the capture of the first label-free target moleculeincludes detecting the first electromagnetic radiation by aphotodetector.
 20. The method of claim 15, wherein the first capturereagent includes a magnetic spin label.